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Growing and Preparing Adult Stem Cells

The question of where exactly in the body stem cells go after they’ve been injected has never been easier to answer, at least in a mouse.

BioInVision has developed an automated system combining a mouse-sized crytotome with a fluorescence (and bright-field) microscope, allowing fluorescently labeled stem cells to be visualized anywhere in the mouse with single-cell sensitivity. The CryoViz™ system will make 40 µm-thick serial cross-sections, “and then we have a microscope that is moved with a robot over the face of the tissue, getting microscopic images of basically the entire mouse,” said David Wilson, Ph.D., BioInVision’s CTO.

Researchers will use the system to look at the biodistribution of stem cells throughout the mouse. Or in the case of myocardial infarction, stem cells may be used to help treat the injury, and CryoViz will be used to evaluate the homing of cells to the infarcted region.

It takes about 15 hours to image an entire mouse. “The stem cell analysis is automated, and it will run by itself. The next day you can have the 3D images,” Dr. Wilson said. “Then if people want counts of cells in different organs, or densities, we have software to make those analyses as well.”

It can be used to image several fluorescent or quantum-dot reporters simultaneously, and it can be combined with vital imaging techniques such as MRI, PET, and bioluminescence. “We use advanced computer algorithms to do the registration … without the need to add extra fiducials (on the arms and legs, for example),” Dr. Wilson said.

The instrument can be purchased from BioInVision or used by means of a fee-for-imaging service, simply by shipping the company the frozen mouse. Using a tape-transfer technology, researchers can also pick up an entire section and treat the fresh-frozen, non-fixed tissue with a histology stain or labeled antibody for immunohistochemistry. “Say in a mouse we do this with 10 or 15 different sections. The advantage is that we know exactly where that section is within the 3D anatomy,” Dr. Wilson said.

Selecting Homing-Ready Stem Cells

Sometimes getting a stem cell to home is a matter of getting it close enough to find the homing beacon. When tissue suffers an ischemic insult—as does the penumbra of tissue surrounding a myocardial infarction—it starts to release the signaling proteins stromal cell-derived factor 1 and vascular endothelial growth factor. These are potent attractors of CD34+, CXCR4+ stem cells, which can help prevent apoptosis, trigger angiogenesis, and preserve the tissue. In the absence of such help, the peri-infarct region is more likely to die in the coming weeks or months.

The problem is that thet stem cells are not hearing the call for help, according to Jonathan Sackner-Bernstein, M.D., vp for clinical development and regulatory affairs at NeoStem.

The company began enrolling patients in January 2012 for Phase II clinical trials of AMR-001, its autologous CD34+, CXCR4+, bone-marrow-derived stem cell product. “We select those cells from the bone marrow, we then administer them into the coronary artery, where they’re smart enough to detect the region of the muscle where they’re needed,” said Dr. Sackner-Bernstein. They move through the walls of the coronary artery into that part of the myocardium and stimulate the development of the blood vessels that the heart is asking for.”

These are cells that are merely selected, not stimulated and expanded in culture. “Once you stimulate cells, we don’t know nearly as much about what they’re going to do and how they’re going to behave when put back into the body,” said Dr. Sackner-Bernstein. “By selecting we’re able to create a product to administer that has a known purity.”

Snag-Free Induction of Pluripotency in Adult Cells

While attractive, the idea of reprogramming adult cells to produce embryonic-like stem cells has not been implemented with much satisfaction. Reprogramming has remained frustratingly slow and inefficient, and it results in stem cells that have limited utility. But now a key impediment to reprogramming has been recognized. Its removal, according to an investigation described recently in Nature (“Deterministic direct reprogramming of somatic cells to pluripotency”), not only shortened reprogramming time by several orders of magnitude, it also improved the efficiency of the process—all the treated cells attained a stem-cell-like state, and they all did so, conveniently, at the same rate.

Adult stem cells may be reprogrammed by inserting four genes into their DNA, a process that yields induced pluripotent stem cells (iPSCs). The process, however, is fraught with difficulty. It can take up to four weeks, the timing is not coordinated among the cells, and one percent or less of the treated cells actually end up becoming stem cells. Success rates can be even lower—around a tenth of a percent—if stem cells are to be used in patients. In such cases, viral gene insertion techniques must be shunned for safety reasons.

The question of efficiency was taken up by researchers at the Weizmann Institute of Science who were already investigating the natural pathways of embryonic development. In particular, researchers in the laboratory of Yaqub Hanna, M.D., Ph.D., asked: What is the main obstacle—or obstacles—preventing successful reprogramming in the majority of cells?

In his post-doctoral research, Dr. Hanna had employed mathematical models to show that a single obstacle was responsible. The identity of the obstacle, however, remained unclear. Then, scientists in Dr. Hanna’s laboratory looked at a certain protein, Mbd3. This protein, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, had caught their attention because it is expressed in every cell in the body, at every stage of development.

Such a protein is quite rare. In general, most types of proteins are produced in specific cells, at specific times, for specific functions. The team found that there is one exception to the rule of universal expression of this protein—the first three days after conception. These are exactly the three days in which the fertilized egg begins dividing, and the nascent embryo is a growing ball of pluripotent stem cells that will eventually supply all the cell types in the body. Starting on the fourth day, differentiation begins and the cells already start to lose their pluripotent status. And that is just when the Mbd3 proteins first appear.

The researchers showed that removing Mbd3 from the adult cells can improve efficiency and speed the process by several orders of magnitude. Efficiency approached 100% from mouse and human cells, and the time needed to produce the stem cells was shortened from four weeks to eight days. As an added bonus, since the cells all underwent the reprogramming at the same rate, the scientists will now be able, for the first time, to actually follow the process step by step and reveal its mechanisms of operation.

Recalling how his team’s discovery was based on research into embryonic development, Dr. Hanna said, “Scientists investigating reprogramming can benefit from a deeper understanding of how embryonic stem cells are produced in nature. After all, nature still makes them best, in the most efficient manner.”

Dr. Hanna’s laboratory is conducting several investigations into iPSCs. These include deciphering the mechanisms of epigenetic reprogramming and induction of pluripotency in somatic cells, including fibroblasts and lymphocytes, as well as the development of iPSC-based experimental systems for in vitro modeling of human disease and development.

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